The ultimate objective of the proposed research is to elucidate transcriptional control mechanisms which may operate through the multiple classes of eukaryotic RNA polymerases themselves. RNA polymerases will be purified from tissues and nuclei at intervals during the modulation (activation) of transcription. Any qualitative changes in enzyme structure (gain or loss of subunits, protein modification, proteolysis, etc.) will be correlated with enzyme activities and in some cases selectivities, as determined by using in vitro reconstituted transcription systems. We will attempt to mimic the changes seen in cellular RNA synthetic rates with the reconstitution systems. In vitro transcription systems will be constructed by using RNA polymerases I, II and III; nuclear templates; and S-100 crude extract (as a source of RNA polymerases I, II and III and as a source of possible accessory transcription factors) from transcriptionally quiescent and active tissues. Various combinations of components from inactive and active systems should indicate, for example, if RNA synthesis rates can be regulated at the level of the RNA polymerase molecule or if gross controls are strictly exerted elsewhere (chromatin structure). Interkingdom reconstituted transcription experiments will be done in order to develop this particular system (wheat germ) as a useful system for in vitro transcription analysis of eukaryotic genetic sequences. Results may indicate how much divergence evolution has allowed in the structure, function and perhaps regulation of the eukaryotic apparatus.